Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

DNA synthesis provides the driving force to accelerate DNA unwinding by a helicase

Nature volume 435, pages 370–373 (2005)Cite this article

Abstract

Helicases are molecular motors that use the energy of nucleoside 5′-triphosphate (NTP) hydrolysis to translocate along a nucleic acid strand and catalyse reactions such as DNA unwinding. The ring-shaped helicase1 of bacteriophage T7 translocates along single-stranded (ss)DNA at a speed of 130 bases per second2; however, T7 helicase slows down nearly tenfold when unwinding the strands of duplex DNA3. Here, we report that T7 DNA polymerase, which is unable to catalyse strand displacement DNA synthesis by itself, can increase the unwinding rate to 114 base pairs per second, bringing the helicase up to similar speeds compared to its translocation along ssDNA. The helicase rate of stimulation depends upon the DNA synthesis rate and does not rely on specific interactions between T7 DNA polymerase and the carboxy-terminal residues of T7 helicase. Efficient duplex DNA synthesis is achieved only by the combined action of the helicase and polymerase. The strand displacement DNA synthesis by the DNA polymerase depends on the unwinding activity of the helicase, which provides ssDNA template. The rapid trapping of the ssDNA bases by the DNA synthesis activity of the polymerase in turn drives the helicase to move forward through duplex DNA at speeds similar to those observed along ssDNA.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: DNA synthesis by T7 DNA polymerase.
Figure 2: The unwinding rate of T7 helicase depends on the speed of DNA synthesis.
Figure 3: DNA synthesis by T7 DNA polymerase and T7 helicase.
Figure 4: DNA synthesis by T4 and T7 DNA polymerases and T7 helicase.

Similar content being viewed by others

References

  1. Patel, S. S. & Picha, K. M. Structure and function of hexameric helicases. Annu. Rev. Biochem. 69, 651–697 (2000)

    Article  CAS  Google Scholar 

  2. Kim, D. E., Narayan, M. & Patel, S. S. T7 DNA helicase: a molecular motor that processively and unidirectionally translocates along single-stranded DNA. J. Mol. Biol. 321, 807–819 (2002)

    Article  CAS  Google Scholar 

  3. Jeong, Y. J., Levin, M. K. & Patel, S. S. The DNA-unwinding mechanism of the ring helicase of bacteriophage T7. Proc. Natl Acad. Sci. USA 101, 7264–7269 (2004)

    Article  ADS  CAS  Google Scholar 

  4. Richardson, C. C. Bacteriophage T7: minimal requirements for the replication of a duplex DNA molecule. Cell 33, 315–317 (1983)

    Article  CAS  Google Scholar 

  5. Lee, J., Chastain, P. D., Kusakabe, T., Griffith, J. D. & Richardson, C. C. Coordinated leading and lagging strand DNA synthesis on a minicircular template. Mol. Cell 1, 1001–1010 (1998)

    Article  CAS  Google Scholar 

  6. Patel, S. S., Wong, I. & Johnson, K. A. Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant. Biochemistry 30, 511–525 (1991)

    Article  CAS  Google Scholar 

  7. Matson, S. W. & Richardson, C. C. DNA-dependent nucleoside 5′-triphosphatase activity of the gene 4 protein of bacteriophage T7. J. Biol. Chem. 258, 14009–14016 (1983)

    CAS  PubMed  Google Scholar 

  8. Matson, S. W., Tabor, S. & Richardson, C. C. The gene 4 protein of bacteriophage T7. Characterization of helicase activity. J. Biol. Chem. 258, 14017–14024 (1983)

    CAS  PubMed  Google Scholar 

  9. Patel, S. S., Rosenberg, A. H., Studier, F. W. & Johnson, K. A. Large scale purification and biochemical characterization of T7 primase/helicase proteins. Evidence for homodimer and heterodimer formation. J. Biol. Chem. 267, 15013–15021 (1992)

    CAS  PubMed  Google Scholar 

  10. Ali, J. A. & Lohman, T. M. Kinetic measurement of the step size of DNA unwinding by Escherichia coli UvrD helicase. Science 275, 377–380 (1997)

    Article  CAS  Google Scholar 

  11. Notarnicola, S. M., Mulcahy, H. L., Lee, J. & Richardson, C. C. The acidic carboxyl terminus of the bacteriophage T7 gene 4 helicase/primase interacts with T7 DNA polymerase. J. Biol. Chem. 272, 18425–18433 (1997)

    Article  CAS  Google Scholar 

  12. von Hippel, P. H. & Delagoutte, E. A general model for nucleic acid helicases and their “coupling” within macromolecular machines. Cell 104, 177–190 (2001)

    Article  CAS  Google Scholar 

  13. Kim, Y. T., Tabor, S., Bortner, C., Griffith, J. D. & Richardson, C. C. Purification and characterization of the bacteriophage T7 gene 2.5 protein. A single-stranded DNA-binding protein. J. Biol. Chem. 267, 15022–15031 (1992)

    CAS  PubMed  Google Scholar 

  14. Lohman, T. M. & Ferrari, M. E. Escherichia coli single-stranded DNA-binding protein: multiple DNA-binding modes and cooperativities. Annu. Rev. Biochem. 63, 527–570 (1994)

    Article  CAS  Google Scholar 

  15. Delagoutte, E. & von Hippel, P. H. Molecular mechanisms of the functional coupling of the helicase (gp41) and polymerase (gp43) of bacteriophage T4 within the DNA replication fork. Biochemistry 40, 4459–4477 (2001)

    Article  CAS  Google Scholar 

  16. Levin, M. K. & Patel, S. S. in Molecular Motors (ed. Schliwa, M.) 179–198 (Wiley, Weinheim, 2003)

    Google Scholar 

  17. Levin, M. K., Gurjar, M. M. & Patel, S. S. ATP binding modulates the nucleic acid affinity of hepatitis C virus helicase. J. Biol. Chem. 278, 23311–23316 (2003)

    Article  CAS  Google Scholar 

  18. Wang, H. & Oster, G. Ratchets, power strokes, and molecular motors. Applied Phys. A 75, 315–323 (2002)

    Article  ADS  CAS  Google Scholar 

  19. Kim, S., Dallmann, H. G., McHenry, C. S. & Marians, K. J. Coupling of a replicative polymerase and helicase: a tau-DnaB interaction mediates rapid replication fork movement. Cell 84, 643–650 (1996)

    Article  CAS  Google Scholar 

  20. Mok, M. & Marians, K. J. The Escherichia coli preprimosome and DNA B helicase can form replication forks that move at the same rate. J. Biol. Chem. 262, 16644–16654 (1987)

    CAS  PubMed  Google Scholar 

  21. Korhonen, J. A., Pham, X. H., Pellegrini, M. & Falkenberg, M. Reconstitution of a minimal mtDNA replisome in vitro. EMBO J. 23, 2423–2429 (2004)

    Article  CAS  Google Scholar 

  22. Schrock, R. D. & Alberts, B. Processivity of the gene 41 DNA helicase at the bacteriophage T4 DNA replication fork. J. Biol. Chem. 271, 16678–16682 (1996)

    Article  CAS  Google Scholar 

  23. Cha, T. A. & Alberts, B. M. The bacteriophage T4 DNA replication fork. Only DNA helicase is required for leading strand DNA synthesis by the DNA polymerase holoenzyme. J. Biol. Chem. 264, 12220–12225 (1989)

    CAS  PubMed  Google Scholar 

  24. Frey, M. W., Nossal, N. G., Capson, T. L. & Benkovic, S. J. Construction and characterization of a bacteriophage T4 DNA polymerase deficient in 3′ → 5′ exonuclease activity. Proc. Natl Acad. Sci. USA 90, 2579–2583 (1993)

    Article  ADS  CAS  Google Scholar 

  25. Lucius, A. L., Maluf, N. K., Fischer, C. J. & Lohman, T. M. General methods for analysis of sequential “n-step” kinetic mechanisms: Application to single turnover kinetics of helicase-catalyzed DNA unwinding. Biophys. J. 85, 2224–2239 (2003)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. O'Donnell, A. Berdis, N. Andraos, C. C. Richardson and M. Salas for the gift of proteins, and C. M. Drain for critical reading of the manuscript. This research was supported by an NIH grant to S.S.P.

Author information

Authors and Affiliations

  1. Department of Biochemistry, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey, 08854, USA

    Natalie M. Stano, Yong-Joo Jeong, Ilker Donmez, Padmaja Tummalapalli & Smita S. Patel

  2. Department of Bio and Nanochemistry, Kookmin University, 861-1, Chongnung-dong, Songbuk-gu, Seoul, 136-702, Korea

    Yong-Joo Jeong

  3. Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, Connecticut, 06030-1507, USA

    Mikhail K. Levin

Authors
  1. Natalie M. Stano
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yong-Joo Jeong
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ilker Donmez
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Padmaja Tummalapalli
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mikhail K. Levin
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Smita S. Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Smita S. Patel.

Ethics declarations

Competing interests

Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

The effect of ssDNA-binding proteins on the unwinding activity of T7 helicase. (JPG 19 kb)

Supplementary Legend

This file contains the legend for Supplementary Fig. 1. (DOC 21 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Stano, N., Jeong, YJ., Donmez, I. et al. DNA synthesis provides the driving force to accelerate DNA unwinding by a helicase. Nature 435, 370–373 (2005). https://doi.org/10.1038/nature03615

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature03615

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing